One of the most fundamental problems in modern biology is to understand dynamic gene activity in time and space in the context of native chromosomes in living cells. The goal of the proposed study is to measure the levels of transcription produced by defined long-range chromosomal interactions in living cells. Traditional live imaging methods lack the spatial resolution to accurately determine the dynamics of gene activity, while bulk assays using fixed material strongly limit investigation of temporal dynamics. Here we propose to overcome these limitations by developing new methods of microscopy and computational analysis. Most of the studies will exploit the unique advantages of the early Drosophila embryo for the development of quantitative live cell imaging methods. Previous studies have identified hundreds of such interactions, and we will sample several of these to provide a titration of varying distances, from tens to hundreds of kilobases, as seen in mammalian systems. There are two specific aims: 1. Develop high-resolution imaging methods and associated computational algorithms for the visualization and quantification of dynamic enhancer-promoter interactions at select endogenous loci in living embryos. 2. Label regulatory regions and associated transcription units of individual genetic loci exhibiting long-range interactions, including trans-homolog associations during transvection at Hox loci, to measure in vivo the effect of chromosome topology on transcriptional activity. We plan to extend this approach to include the visualization of several hundred fluorescent DNA foci in a library of genetically engineered fly lines to establish a general overview of the dynamics of an entire chromosome in a living embryo and its impact on transcription. The successful realization of the proposed studies will greatly augment our current capacity to superimpose whole-genome maps based on fixed tissues onto the dynamic chromosomes of living cells. The resulting technologies will be immediately applied to the visualization of chromosome dynamics in mammalian tissues, particularly multipotent progenitor cells such as mouse hepatoblasts.